Sequencing of DNA is a major driver in genetics research. The ‘next generation sequencing’ technological revolution is gathering momentum as new robust high-throughput sequencing instruments are becoming available. New and improved methods and protocols have been developed to support a diverse range of applications, including analysis of genetic variation. As part of this, methods have been developed that aim to achieve targeted enrichment of genome sub-regions such as targeted cancer panels or complete human exomes. By selective recovery of genomic loci of interest, costs and effort can be reduced significantly compared with whole-genome sequencing.
Current techniques for targeted enrichment fall into three categories; Hybrid capture, selective circularization, and PCR amplification. In hybrid capture techniques, short fragment libraries (typically 100-250 base pairs) are hybridized specifically to complementary DNA fragments so that one can physically capture and isolate the sequences of interest. Selective circularization encompasses methods wherein single-stranded DNA circles including target sequences are formed, creating structures with common DNA elements that are then used for selective amplification of the target sequence. Finally, PCR amplification based enrichment is directed toward the target region by conducting multiple PCR reactions in parallel.
Common for the current enrichment methods is that they require a significant knowledge of the target sequence, a relatively pure sample, a significant amount of target sequence, and that they produce relatively short sequences.
Accordingly there is a need for methods that can be used to analyze complex DNA samples, for example mammalian genomic DNA samples, where the frequency of the target is low (i.e. 1 target copy per genome), and where the analysis does not depend on the individualized analysis of each DNA molecule in the sample, which is both expensive and time consuming.